The Expression, Purification And Activity Analysis Of Snake Metalloproteinases Inhibitor (BJ46a) In Pichia Pastoris

BJ46a is a protein inhibitor of the snake venom metalloproteinases(SVMPs) protelytic activities. It is from the serum of the venomous snake Bothrops jararaca. Being a new member of the fetuin family, cystatin superfamily, BJ46a interacts with the metalloproteinases by the formation of a noncovalent complex which occurs between the metalloproteinase domain and the inhibitor. Both SVMPs and MMPs protein families belong to the superfamily metzincin of metalloproteinases, so BJ46a may inhibit tumor invasion and metastasis by the formation of a complex with MMPs. In this study, BJ46a recombinant protein was produce by pichia pastoris expression system, then assay its activity, and make preperation for BJ46a as a new drug of antitumor invision and metastasis in the future.1. Construction of pichia pastoris expression vector for BJ46a geneBy using of molecular cloning technology, DNA fragment of BJ46a encoding gene(no including signal peptide) was amplified by PCR from Bacmid-BJ46a plasmid and then inserted into pPICZαA vector down theα-factor signal sequence to construct an expression plasmid pPICZαA- BJ46a..2. Expression and purification of BJ46a in Pichia Pastoris expression systemThe constructed pPICZαA-BJ46a was linearized by SacⅠand transformed into yeast Pichia pastoris, strain GS115 by the electrophoration. A quick ,direct way to select putative multi-copy recombinants is to plate the transformation mix on increasing concentrations of zeocinTM. The positive clones were identified by PCR analysis. GS115-BJ46a transformants were induced in 1.0% methanol and the secretion expression protein was examined by SDS-PAGE and Western blot analysis. The molecular mass of experssion product was about 38kDa. About 10 mg/L pure BJ46a was obtained after purified by ProBond protein purification system.3. Determination physico-chemical property of recombination protein BJ46a and activity analysis physico-chemical property of recombination protein BJ46a including: determine PI≈5.8 through isoelectric focusing electrophoresis; purity determination is100% with HPLC; molecular mass identification is 38754Da by MS. Purified BJ46a protein was assayed for biological activity and the result indicated that BJ46a could inhibit the activity of MMPs.In conclusion,this study provides the evidence that recombination protein BJ46a can be obtained by Pichia Pastoris expression system and indicated that it could inhibit the activity of MMPs.